The present invention relates generally to vectors and methods for introducing genetic material into an embryo of a chicken and other avian species and, more particularly, to vectors and methods for transferring a gene of interest to an embryonic chicken cell, so as to create a transgenic hen having the gene of interest expressed in the hen""s oviduct and the gene product secreted in the hen""s eggs and/or those of her offspring.
Since the development of recombinant DNA technology some twenty-five years ago, the prospect of producing proteins on a large scale, rather than extracting them from tissue where they are naturally expressed, has become a reality. In particular, over the last two decades, progress in the development of expression vectors has led to the production of thousands of recombinant proteins on a laboratory scale. Production of commercial quantities of recombinant proteins requires often difficult and expensive scaling up procedures, but has nonetheless also been successful. In addition, transgenic animals including mice, rabbits, pigs, sheep, goats and cows have been engineered to produce human pharmaceuticals in their tissues or secretions. Houdebine, L. M., J. Biotechnology 34:269-287 (1994).
Although egg white is thought to be an excellent host for recombinant protein production, preparing transgenic avians has proven to be technically difficult due in large part to problems involved in manipulating the chicken embryo. When oviposition occurs, the embryo has already reached a stage corresponding to a mammalian late blastula or early gastrula. Genetic manipulation of the embryo during earlier development requires reintroduction to the female or in vitro culture, both difficult procedures. Houdebine, L. M., J. Biotechnology 34:269-287 (1994). Despite these difficulties, transgenic chickens have been produced that are resistant to infection by avian leukosis (Crittenden and Salter, xe2x80x9cTransgenic Livestock Models In Medicine And Agriculturexe2x80x9d pp. 73-87 (Wiley-Liss (1990))), or have high levels of circulating growth hormone. Bosselman, R. A., et al., Science 243:533-535 (1989).
Four general methods for generating transgenic avians have been reported. One method involves excision of a developing egg from the oviduct, microinjection of DNA near the blastoderm, and in vitro culture of the manipulated embryo in solution and surrogate shells. Love, J., et al., Biotechnology 12:60-63 (1994). A second method requires the culture and transfection of primordial germ cells, with subsequent transplantation into an irradiated recipient near the same stage of development as the donor. Carsience et al., Development 117:669-675 (1993); Etches et al., Poultry Science 72:882-889 (1993). Although technically very demanding, these two approaches are attractive because large pieces of DNA can be transferred.
A third method involves blind injection of replication competent retrovirus with a needle near the blastoderm of a newly laid egg. Petropoulos, C. J., et al., J. Virol. 65:3728-3737 (1991). Although this method is the simplest, it is also limited in that the DNA to be transferred must be approximately 2 kb or less in size and, the method results in viremic hens which shed infective recombinant retrovirus. Petropoulos, C. J., et al., J. Virol. 66(6):3391-3397 (1992).
The fourth method involves a replication-defective retroviral vector system (see, e.g., U.S. Pat. Nos. 5,162,215 and 4,650,764, hereby incorporated by reference). One of these systems (Watanabe and Temin, Mol. Cell. Biol. 3(12):2241-2249 (1983)) has been derived from the reticuloendotheliosis virus type A (REV-A). Sevoian et al., Avian Dis. 8:336-347 (1964). Replication-defective retroviral vectors derived from the REV-A virus are based on the helper cell line C3 (Watanabe and Temin, Mol. Cell. Biol. 3(12):2241-2249 (1983)) which contains the components of a packaging defective helper provirus. The derivation of the C3 helping line and several replication-defective retroviral vectors have been described in detail in U.S. Pat. No. 4,650,764 and Watanabe and Temin, Mol. Cell Biol. 3(12):2241-2249 (1983). This method is more technically demanding than the replication competent technique in that the blastoderm must be exposed, and microinjection equipment must be used. Bosselman, R. A., et al., Science 243:533-535 (1989). Nonetheless, it results in transgenic hens free of replication competent retrovirus, and can transfer DNA as large as 8 kb in size.
Tissue specific expression of a foreign gene in a transgenic chicken was achieved using the replication competent retrovirus technique. Petropoulos, C. J., et al., J. Virol. 66(6):3391-3397 (1992). A replication competent retrovirus was used to deliver the reporter gene chloramphenicol acetyl transferase (CAT), driven by a muscle specific promoter, a action, to skeletal muscle. Tissue specific expression of a recombinant protein in the egg of a transgenic avian has not yet been successful.
It would thus be desirable to provide a vehicle and method for transferring a gene to an embryonic chicken cell (or other avian species) so as to create a transgenic hen wherein the gene is expressed in a tissue specific manner. It would also be desirable to provide a vehicle and method for transferring a gene to an embryonic chicken cell, wherein the gene is expressed in the hen""s oviduct and secretion of the gene product is in the hen""s eggs. It would also be desirable to provide a vehicle and method for transferring a gene to an embryonic chicken cell, wherein the gene is expressed in the hen""s oviduct and secretion of the gene product is in the hen""s eggs without compromise to the hen""s health and the health of other birds in contact with her.
Vectors and methods are provided for introducing genetic material into cells of a chicken or other avian species. More particularly, vectors and methods are provided for transferring a transgene to an embryonic chicken cell, so as to create a transgenic hen wherein the transgene is expressed in the hen""s oviduct and the transgene product is secreted in the hen""s eggs and/or those of her offspring. In a preferred embodiment, the transgene product is secreted in the egg white.
In one embodiment, the vector comprises a portion of a retroviral genome, capable of transfecting a cell and incapable of replication, i.e., a replication-defective retroviral vector. The vector further comprises a transgene, operatively-linked to appropriate control elements such that the transgene may be expressed in a tissue specific manner. In one embodiment, the control elements include an enhanced promoter directing the expression of the transgene in the oviduct, an untranslated region 5xe2x80x2 to the structural gene (coding region) of appropriate length and sequence to promote efficient translation, and a signal sequence directing the secretion of the transgene product in the egg white. In this embodiment, the promoter may be chosen, without limitation, from the group consisting of ovalbumin, lysozyme, conalbumin and ovomucoid promoters, and combinations thereof. In another embodiment, the control sequences include a promoter directing the expression of the transgene in the liver and a signal sequence directing the uptake and secretion of the transgene product into the egg yolk. In this embodiment, the promoter may be chosen, without limitation, from the group consisting of vitellogenin and apolipoprotein A promoters, and combinations thereof.
The vectors of the present invention may be used in producing transgenic avians, particularly chickens, by methods known to those skilled in the art, such as the four methods described above (see, Background Of The Invention). For example, as described in U.S. Pat. No. 5,162,215, herein incorporated by reference, the vectors may be used to introduce a nucleic acid sequence, e.g., a gene, into germ cells and stem cells of an embryo of a chicken. In one embodiment, a retrovirus generated from the vector is microinjected in a newly laid chicken egg, in close proximity to (e.g., directly beneath) the blastoderm The egg is then sealed and incubated until the chicken is hatched from the egg. The chimeric chicken is then tested for expression of the transgene and if positive and the chicken is female (hen), the eggs of the chicken arc harvested and the protein is isolated by methods known to those skilled in the art. If the chicken is male (rooster), it can be bred to produce a female transgenic chicken whose eggs may then be harvested. Transgenic avians and eggs, as well as methods of making transgenic avians and eggs, are thus provided.
Other features and advantages of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.